Gibberellins (GAs) form a large family of tetracyclic diterpenoid carboxylic acids that have the basic structure called ent-gibberellane (FIG. 1A). They control multiple processes in the life cycle of higher plants, which are essential for normal plant growth and development (Graebe, J. E. (1987). Ann. Rev. Plant Physiol., 38, 419–465; Hooley, R. (1994). Plant Mol. Biol., 26, 1529–1555). Biologically active GAs, such as GA1, are produced from trans-geranylgeranyl diphosphate mediated by the sequential actions of cyclases in the plastids, membrane-associated monooxygenases at the endoplasmic reticulum and soluble 2-oxoglutarate-dependent dioxygenases located within the cytosol (reviewed in Hedden, P. and Kamiya, Y. (1997). Ann. Rev. Plant Physiol. Plant Mol. Biol., 48, 431–460; Lange, T. (1998). Planta, 204, 409–419). The biosynthetic pathway of GA is well established (FIG. 1B). The 2-oxoglutarate-dependent dioxygenases catalyze the later steps in the biosynthetic pathway, including the removal of C-20 by GA 20-oxidase and the introduction of the 3β-hydroxyl group by GA 3β-hydroxylase to synthesize biologically active GAs. A third dioxygenase, GA 2β-hydroxylase, introduces a 2β-hydroxyl group resulting in biologically inactive GAs that cannot be converted into active forms.
In recent years, cDNAs and genomic clones encoding GA biosynthetic enzymes have been isolated from various plant species (reviewed in Hedden, P. and Kamiya, Y. (1997). Ann. Rev. Plant Physiol. Plant Mol. Biol., 48, 431–460; Lange, T. (1998). Planta, 204, 409–419). Availability of these clones has been clarifying the regulation of GA biosynthesis. The GA 20-oxidases, for example, are shown to be encoded by several genes that are differentially regulated throughout plant development (Phillips, A. L. et al. (1995). Plant Physiol., 108, 1049–1057; Garcia-Martinez, J. L. et al. (1997). Plant Mol. Biol., 33, 1073–1084). Although GA 2β-hydroxylases play an important role in determining the endogenous concentration of bioactive GAs, the genes for these enzymes have not been isolated until recently. The first isolation of GA 2β-hydroxylase genes was from scarlet runner bean (Phaseolus coccineus L.) and Arabidopsis thaliana by a functional screening method (Thomas, S. G. et al. (1999). Proc. Natl. Acad. Sci. USA, 96, 4698–4703).
GAs are involved in many developmental processes, including germination, stem elongation, flowering, and fruit development. Therefore, modifications of these processes by application of chemicals that alter GA content are common agronomic and horticultural practices. For instance, GA3 is used to stimulate berry growth in seedless grape production (Christadoulou, A. J. et al. (1968) Proc. Am. Soc. Hort. Sci., 92, 301–310), and GA biosynthesis inhibitors are used as growth retardants to control the height of cereal crops and ornamental plants (Hedden, P. and Hoad, G. (1994). Growth regulators and crop productivity. Mechanisms of Plant Growth and Improved Productivity: Modern Approaches (Basra, A. S., ed.). New York: Marcel Dekker, pp. 173–198). An alternative approach to the exogenous application of chemicals would be to modify the endogenous content of GAs via genetic manipulation of their biosynthesis. The recent cloning of several genes involved in GA biosynthesis provided the means to test the feasibility of this approach. Isolation of genes encoding GA 2β-hydroxylase was, in particular, expected to bring a powerful tool to control the bioactive GA content in transgenic plants.
A number of GA-responsive mutants have been isolated from various plant species, such as maize, pea, tomato, Arabidopsis, and rice (Phinney, B. O. (1956). Proc. Natl. Acad. Sci. USA, 42, 185–189; Koornneef, M. (1978). Arabidopsis Ins. Serv., 15, 17–20; Koornneef, M. et al. (1990). Theor. Appl. Genet., 80, 852–857; Reid, J. B. and Ross, J. J. (1993). Int. J. Plant Sci., 154, 22–34; Murakami, Y. (1972). Plant Growth Substances 1970. (Carr, D. J. ed.) Berlin: Splinger-Verlag, pp. 166–174). Phenotypes resulting from reduced GA production in spontaneous mutants of Arabidopsis imply the role of GAs in stem elongation and flowering (Koornneef, M. and van der Veen, J. H. (1980). Theor. Appl. Genet., 58, 257–263; Sponsel, V. M. et al. (1997). Plant Physiol., 115, 1009–1020). GA1 encodes copalyl-diphosphate synthase (CPS). Null mutations in this locus inhibit stem elongation in long day conditions to cause flowering without bolting and both stem elongation and flowering in short day conditions (Wilson, R. N. et al. (1992). Plant Physiol., 100, 403–408; Sun, T. -P. and Kamiya, Y. (1994). Plant Cell, 6, 1509–1518). GA4 encodes GA 3β-hydroxylase (Chiang, H. -H. et al. (1995). Plant Cell, 7, 195–201; Williams, J. et al. (1998). Plant Physiol., 117, 559–563), while GA5 and GA6 encode distinct GA 20-oxidases (Xu, Y. L. et al. (1995). Proc. Natl. Acad. Sci. USA., 92, 6640–6644; Sponsel, V. M. et al. (1997). Plant Physiol., 115, 1009–1020). Null mutations in both GA4 and GA5 result in semi-dwarfs with normal flower development. In contrast, loss of function of GA6 results in short inflorescences, reduced fertility and short siliques (Sponsel, V. M. et al. (1997). Plant Physiol., 115, 1009–1020).
GA-deficient mutants have also been isolated from rice (dx: d35 and dy: d18). The rice dwarf mutants have considerable agricultural significance. For example, sd-1 mutants are especially important for rice breeding because they are the genetic basis of high yielding, semi-dwarf varieties. Rice d18 mutants are GA responsive dwarf, and multiple alleles have been identified; Housetu-waisei (d18h), Akibare-waisei (d18-AD), Kotake- tamanishiki (d18k), and Waito-C (d18-w) were isolated from different parental ecotypes. The analyses of GA intermediates in d18 mutants demonstrated that the conversion to 3β-hydroxyl GAs was blocked in these mutants. This resulted in the accumulation of endogenous level of GA20 and the drastic decrease of bioactive GA1 content (Kobayashi, M. et al. (1988). Plant Cell Physiol., 30(7): 963–969; Kobayashi, M. et al. (1994). Plant Physiol. 106: 1367–1372; Choi Y-H. et al., (1995). Plant Cell Physiol. 36(6): 997–1001). These findings strongly suggest that the D18 gene encodes a GA 3β-hydroxylase and reduction of GA1 suppresses stem elongation in mutant plants.
The dwarf stature characteristic is one of the most valuable traits for breeding of agricultural and horticultural crops including fruit trees because this feature enables high density planting, efficient reception of light, decrease of wind damage, and reduction of farming labor. It is possible to reduce endogenous levels of bioactive GAs in transgenic plants. For example, antisense expression of Arabidopsis GA 20-oxidase gene and tobacco GA 3β-hydroxylase gene decreases the level of active GAs, and results in semi-dwarf phenotypes (Coles et al., (1999). Plant. J. 17, 547–556; Itoh et al., (1999). Plant. J. 20, 15–24). However, this method of producing dwarf plants by antisense expression of these active GA-forming enzyme genes has two major defects: 1) it is difficult to predict and regulate an endogenous level of GA, because expression of homologue genes which exist in the same species as the plant, into which the antisense construct will be introduced, may not be suppressed and the half-life of active gibberellins is extended due to the suppression of expression of genes encoding 2β-hydroxydases that produces biologically inactive GAs, and 2) it is necessary to isolate the corresponding cDNA from the same plant species as the plant into which the antisense construct will be introduced.
In contrast, since the structure of active GAs, which are substrates for GA deactivation enzymes, is preserved in other plants, overexpression of GA deactivation enzyme genes, such as GA 2β-hydroxylase gene, is probably effective in heterologous plant species. Moreover, it could easily regulate the active GA content to a preferable level via modification of transgene expression.